SUMMARY Our research goal is to determine the regulatory mechanisms controlling the function of Ewing sarcoma fusion proteins during induction of mitotic dysfunction, chromosomal instability (CIN), and ultimately in the pathogenesis of Ewing sarcoma, a pediatric bone cancer. Ewing sarcoma cells express aberrant EWS/FLI1 fusion protein, derived by chromosomal translocation, and this leads to impaired target gene expression through altered transcription and splicing. In addition to these mechanisms, we previously identified a novel function of EWS/FLI1 which leads to mitotic dysfunction through interaction with EWS and inhibition of EWS activity. Our study demonstrated that EWS regulates mitosis through interaction with Aurora B kinase (Aurora B), and through recruitment of Aurora B to the midzone, a midline structure that is located between segregating chromosomes. Disruption of EWS-Aurora B interaction by EWS/FLI1 protein may be a contributing factor in the development of Ewing sarcoma, because compromised Aurora B-relocation leads to defects in cytokinesis, missegregation of chromosomes, and ultimately, to induction of CIN. The mechanisms that modulate EWS/FLI1 activity during the induction of mitotic dysfunction and CIN are unknown. One possible regulatory mechanism is post-translational modification (PTM), and a previous study showed that EWS and EWS/FLI1 undergo O-GlcNAcylation, a form of PTM wherein a single ?-N- acetylglucosamine sugar is attached to Ser or Thr residues. We therefore chose to investigate O-GlcNAcylation as a potential regulatory mechanism for EWS/FLI1-dependent mitotic dysfunction. Our previous study demonstrated that elevated cellular O-GlcNAc levels induce midzone formation defects in HeLa cells. Because EWS forms homodimers, and EWS/FLI1 interaction with EWS disrupts its mitotic function, we hypothesize that O-GlcNAcylation of EWS/FLI1 induces midzone dysfunction by enabling the fusion protein to form a stable dimer with EWS. We will utilize an EWS/FLI1 construct containing a point mutation at the proposed critical O-GlcNAcylation site in human cells (HeLa and Ewing sarcoma A673 cells), to investigate our hypothesis, by addressing the following specific aim. Specific Aim 1: To determine how O-GlcNAcylation of EWS/FLI1 and EWS affects the induction of CIN. In addition to EWS/FLI1 in Ewing sarcoma, EWS is fused to different genes in other types of sarcomas, and the resulting EWS-fusion genes are thought to be responsible for transformation. The significance of this study is that impairment of mitosis by O-GlcNAcylation of EWS-fusion proteins may be a universal mechanism for pathogenesis in all sarcomas expressing EWS-fusion genes. Thus, our study may supply a platform for understanding the molecular mechanism regulating all EWS-fusion gene derived sarcomas. 1